Hereditary NonPolyposis Colorectal Cancer (HNPCC) is one of the most frequent cancer predisposition syndromes and is characterized by early onset of colorectal cancers as well as cancers of the endometrium, ovary, skin, stomach and upper urinary tract. Germline mutations in two of the seven mitotic human mismatch repair (MMR) related genes, hMSH2 and hMLH 1, account for the vast majority of HNPCC cases, while mutations in the hMSH6 and hPMS2 MMR genes are rare. In addition, 10-40% of sporadic tumors display the genetic instability (mutator phenotype) that is a hallmark of human mismatch repair defects. Since our initial discovery of hMSH2 and hMLH1, we have arguably accumulated the most complete repertoire of MMR and MMR-related genes/proteins/reagents. In the last grant period we interrogated the biochemistry, genetics, cell and tumor biology of human MMR. We have detailed a novel mechanism for MMR, the functional consequences of hMSH2 HNPCC missense mutations, and revised the causes and consequences of MMR mutations in the process of carcinogenesis. In this continuing application we propose to: I.) perform the first complete biophysical and proteogenetic characterization of mismatch/lesion recognition and ATP-processing ofMutS homologs (MSH) in any organism using the human protein heterodimers as a model, II.) biophysical and proteo-genetic characterization of the interactions and ATP processing activities of the human MutL homologs (MLH) with ATP-bound human MSH sliding clamps, IlI.) examine the interaction and excision mechanics of MSH/MLH signaling sliding clamps with the BLM helicase, ExoI, and PCNA, IV.) determine the functional defects of HNPCC missense mutations on MLH, hExoI, and PCNA biochemical function in MMR, and V.) gradual reconstitution of the entire MMR signaling and excision-repair process in order to characterize the nanoscale biophysical events associated with the complete repair reaction. These studies will provide the molecular and functional roles of the human MMR components, detail the signaling processes that contribute to HNPCC, and assist in the identification of clear molecular targets for functional therapeutic intervention and increased clinical efficacy in human cancer.